Osteopontin (OPN) is a secreted phosphoprotein that has been implicated in the development and progression of several solid tumors. In the previous project, we have identified OPN as a secreted marker for tumor hypoxia and a powerful prognostic marker for head and neck (H&N) cancers. It is therefore a natural progression for this project to focus on the role of OPN on tumor progression in H&N cancers. The major goal of this project is to systematically investigate the effect of OPN and its individual domains on tumor invasion and metastasis in H&N cancers and to explore the possibility of targeting OPN using OPN blocking antibodies as a novel anticancer therapy. Specifically, we will generate stable cell lines expressing full-length OPN, its individual domains or its mutant constructs under a tetracycline-regulatable promoter in an H&N cancer cell line that is known to have minimal basal OPN expression. We will determine the impact of OPN and its domains on cell invasion and metastasis under normoxia and hypoxia in vitro and in vivo. We will use molecular tools to explore the mechanisms by which OPN enhances tumor invasion in H&N cancers. At the same time, we will evaluate the impact of blocking the expression OPN and its domains using domain- specific antibodies (abs) on tumor invasion, growth and metastasis in nude-mouse xenografts and to explore the use of OPN blocking abs for novel therapy. Dinitrobenzamide mustards (DNBMs) constitute a new and highly potent class of hypoxic cytotoxins that will be studied in detail in Project 2. Since we have found that OPN significantly enhances H&N cancer cell invasion and metastasis under hypoxia, we hypothesize that targeting both OPN and hypoxia may be better than targeting individual component alone. Therefore, the last specific aim of the project will evaluate the efficacy of combining OPN abs with PR-104 + radiation on tumor growth and metastasis in comparison to each treatment alone. In summary, the proposed aims are intended to answer the fundamental questions about the mechanisms by which OPN enhances the malignant tumor phenotype in H&N cancers and to identify the optimal approach to target this protein for clinical use.